The overall objective of this research is to understand the role of protein phosphatases in the mechanisms of hormonal signal transduction. Hormones such as epinephrine and insulin elicit their physiological responses by altering the phosphorylation state of key regulatory intracellular phosphoproteins and protein phosphatases are almost certainly involved in these controls. This project addresses one of the main classes of protein phosphatases, the type 1 phosphatases, and in particular, two specific enzymes termed the ATP-Mg-dependent phosphatase and the glycogen-bound phosphatase. Knowledge of the function and regulation of these enzymes is incomplete. Both enzymes appear to be controlled by multisite phosphorylation of their regulatory subunits. Structure-function studies will be aimed at identifying and understanding the roles of the individual phosphorylation sites on the two regulatory for interactions between the regulatory and catalytic components will also be sought. Methods to be used include site-directed mutagenesis and deletion mutations. In addition, competition studies utilizing synthetic peptides and specific antibody probes will be employed. Hormonal regulation studies will be employed. Hormonal regulation studies will utilize diaphragm muscle systems and cultured cells and will focus on the action of insulin and epinephrine. Expression in mammalian cultured cell lines of inhibitor-2 and G-subunit mutated at the individual phosphorylation sites should help probe the physiological role of each site and the mechanisms mediating transmission of the hormone signals. Success in this investigation will be of widespread significance since these phosphatases are believed to regulate a variety of metabolic pathways and other cellular functions.